The long-term goal of work outlined in this proposal is to characterize factors affecting the activation of carcinogens via mixed-function oxidase systems in intact cells. Initial studies will be carried out using the isolated hemoglobin-free perfused rat liver as a model tissue in which the production of oxidized drug metabolites will be continuously monitored and correlated with changes in steady-state levels of key intermediates of energy metabolism and with the oxidation-reduction state of pyridine nucleotides, flavins and cytochromes measured by tissue surface fluorescence and absorbancy. Rates of mixed-function oxidation will be experimentally manipulated by exposure of animals to inducing agents, metabolic inhibitors and altered nutritional states. Information gathered in these studies will be used to identify metabolic events associated with the regulation of rates of mixed-function oxidation in intact hepatocytes. In order to gain insight into mechanisms generating NADPH, an obligatory substrate for mixed-function oxidation, substrates in equilibrium with this cofactor will be measured in extracts of livers freeze-clamped during the course of mixed-function oxidation. In addition, the effect of inducing agents and dietary alterations on enzyme activities associated with the generation of NADPH will be examined. During the course of this work methods will be developed to continuously monitor the metabolism of several carcinogens in perfused organs and experiments will be conducted to describe interactions between intermediary metabolism and carcinogen activation in several tissues, including rat liver, livers from mice which differ in their susceptibility to selected carcinogens, and from lung. Knowledge gained in these studies may be ultimately applied to experimentally manipulating the incidence and rate of chemical carcinogenesis in laboratory animals.